The broad objective of this project is the detailed molecular characterization of the Na+/I- symporter (NIS) a key intrinsic plasma membrane transport protein that mediates the active translocation of I- in the thyroid. NIS plays a crucial role in thyroid hormogenesis and in the evaluation, diagnosis, and treatment of thyroid diseases, including cancer. Earlier in this project, the NIS cDNA was isolated, resulting in remarkable progress in the molecular characterization of NIS. The biogenesis and post-translational modifications of NIS were elucidated, a 2:1 Na+/I- transport stoichiometry and an ordered ionic binding mechanism were demonstrated by electrophysiological analysis, and the regulation of NIS transcription was determined. The roles of functionally important amino acid residues in NIS have been identified by characterizing NIS mutations that cause congenital I- transport defect. NIS also mediates iodide transport in very few other tissues, including salivary glands, gastric mucosa and lactating breast and in breast cancer. NIS expression in breast cancer is of potential diagnostic and therapeutic significance. To reach the broad objective of this project, the following specific aims will be pursued: 1. To elucidate regulatory mechanisms of NIS, by examining the roles of: a) NIS phosphorylation in NIS trafficking and activity; b) TSH in modulating the rates of NIS targeting to the plasma membrane and/or internalization; c) the PDZ motif in the polarized targeting of NIS to the basolateral membrane; and d) the proteasome in the regulation of NIS targeting to the plasma membrane, trafficking, and degradation. 2. To establish structure-function relations in NIS, by a) extending the analysis of NIS topology; b) performing an electrophysiological analysis of mutant NIS proteins to assess the role of functionally important amino acid residues in the Na+ and I- translocation pathways, as well as in Na+/I- coupling; c) identifying amino acid side chains that are modified by the NIS inhibitor perchlorate; d) optimizing conditions for NIS solubilization, reconstitution and purification, thus making future structural studies possible. 3. To assess NIS expression in the stomach under physiological and pathophysiological conditions by addressing the differences in NIS expression among different regions of the stomach, in different types of gastric cells, and in premalignant gastric lesions. The continued molecular analysis of NIS clearly holds the potential of an even greater impact on a wide spectrum of fields, ranging from structure-function of transport proteins to the diagnosis and treatment of cancer, both in the thyroid and beyond.